System and method of biocatalytic conversion for production of alcohols, ketones, and organic acids

ABSTRACT

Biocatalytic conversion systems and methods of producing and using same that have improved yields are disclosed. The systems and methods involve co-fermentation of sugars and gaseous substrates for alcohol, ketone, and/or organic acid production. The systems and methods may include biocatalytically converting at least one sugar substrate into at least one of alcohol, at least one ketone, and/or at least one organic acid. The systems and methods may further include biocatalytically converting gases that comprise CO2 and H2 to at least one alcohol and/or at least one organic acid, thereby adding extra revenue to biorefineries.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U. S. Ser. No.16/800,541, filed Feb. 25, 2020, which is a continuation application ofU. S. Ser. No. 16/604,242, filed Oct. 10, 2019, now issued as U. S.patent Ser. No. 11/180,779, which is a US National Stage Applicationunder 35 USC § 371 of International Application No. PCT/US2018/27285,filed Apr. 12, 2018, which claims benefit under 35 USC § 119(e) of U. S.Provisional Patent Application No. 62/484,525, filed on Apr. 12, 2017.The entire contents of the above-referenced application are herebyexpressly incorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U. S. Government support under DOT GrantNo. DTOS59-07-G-00053 awarded by the Department of Transportation andis/was supported by the USDA National Institute of Food and Agriculture(NIFA) Award No. 2019-38502-30120/South Dakota State University SubawardNo. 3TC386. The Government has certain rights in the invention.

BACKGROUND

Butanol is a drop-in fuel, which in many cases is more desirable thanethanol due to its higher energy density and compatibility with existingfuel infrastructure (Fortman et al., 2008; Wang et al., 2009). Butanolcan be converted with a hydrogenation step to drop-in diesel and jetfuels (Simmons, 2011; Yang and Wyman, 2008).

Butanol has been produced by the traditional acetone-butanol-ethanol(ABE) fermentation using molasses and starches (Jones and Woods, 1986;Ni and Sun, 2009), and recently from lignocellulosic biomass (Liu etal., 2015a; Liu et al., 2015b; Qureshi et al., 2010). Unfortunately, theyield of butanol from biomass conversion is poor because a large amountof the biomass is used by solventogenic Clostridium species for theproduction of un-captured CO₂ and H₂. During butanol production via ABEfermentation, more than 50% of the carbon from sugars is wasted inproducing H₂ and CO₂ (Zhu and Yang, 2010). Most of these gas byproductsare released into the atmosphere, leaving a negative impact on processeconomy. Therefore, it is critical to improve ABE yields from renewablesources to make this process viable.

Thus, the present disclosure is directed to new and improvedbiocatalytic conversion systems and methods of producing and using samethat have improved yields and thus overcome the disadvantages anddefects of the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an embodiment of a co-fermentationsystem in two stage reactor and co-culture in one stage reactor with anexample of bacteria (Ca: Clostridium acetobutylicum and Cr: Clostridiumragsdalei) and possible products that can be made. ABE:acetone-butanol-ethanol; EtOH: ethanol; i-PrOH: isopropanol; BuOH:butanol; HAc: acetic acid; and HBu: butyric acid.

FIG. 2 contains an exemplary schematic diagram of co-fermentation ofsugars in Fermentor A and CO₂ and H₂ in Fermentor B. 1: valves forsampling liquid; 2: gas line; and 3: rubber stopper for sealing and gassampling.

FIG. 3 graphically illustrates (▴) Growth and pH, (B) glucose, (C)solvents, and (D) organic acids profiles during ABE fermentation inFermentor A using C. acetobutylicum ATCC 824 with Medium I (pureglucose) (n=3).

FIG. 4 graphically illustrates (▴) Growth and pH, (B) gasconsumption/production, (C) ethanol and acetic acid, and (D) absoluteheadspace pressure profiles during gas fermentation in Fermentor B usingC. ragsdalei with Medium I (pure glucose) in Fermentor A (n=3).

FIG. 5 graphically illustrates (▴) Growth and pH, (B) glucose, (C)solvents, and (D) organic acids profiles during ABE fermentation inFermentor A using C. acetobutylicum ATCC 824 with Medium II (redcedarhydrolyzate) (n=3) for an embodiment.

FIG. 6 graphically illustrates (▴) Growth and pH, (B) gasconsumption/production, (C) ethanol and acetic acid, and (D) absoluteheadspace pressure profiles during gas fermentation in Fermentor B usingC. ragsdalei with Medium II (redcedar hydrolyzate) in Fermentor A (n=3)for an embodiment.

FIG. 7 graphically illustrates growth and acetic acid and ethanolproduction during conversion of CO₂ by (∘) Clostridium ragsdalei, (▴)Clostridium carboxidivorans, and (□) Clostridium muellerianum.

FIG. 8 graphically illustrates butyric acid and butanol productionprofiles during conversion of CO₂ by (∘) Clostridium ragsdalei, (▴)Clostridium carboxidivorans, and (□) Clostridium muellerianum.

FIG. 9 graphically illustrates hexanoic acid and hexanol productionprofiles during conversion of CO₂ by (∘) Clostridium ragsdalei, (▴)Clostridium carboxidivorans, and (□) Clostridium muellerianum.

FIG. 10 graphically illustrates cumulative CO₂ and H₂ uptake rateprofiles during conversion of CO₂ (∘) Clostridium ragsdalei, (▴)Clostridium carboxidivorans, and (□) Clostridium muellerianum.

DETAILED DESCRIPTION

Before explaining at least one embodiment of the inventive concept(s) indetail by way of exemplary language and results, it is to be understoodthat the inventive concept(s) is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description. The inventive concept(s) is capable ofother embodiments or of being practiced or carried out in various ways.As such, the language used herein is intended to be given the broadestpossible scope and meaning; and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed inventive concept(s) shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses and chemical analyses.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this presently disclosed inventiveconcept(s) pertains. All patents, published patent applications, andnon-patent publications referenced in any portion of this applicationare herein expressly incorporated by reference in their entirety to thesame extent as if each individual patent or publication was specificallyand individually indicated to be incorporated by reference.

All of the compositions and/or methods disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of the inventiveconcept(s) have been described in terms of particular embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit, and scope of the inventive concept(s). All suchsimilar substitutions and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinventive concept(s) as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the term “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” As such, the terms “a,” “an,” and “the”include plural referents unless the context clearly indicates otherwise.Thus, for example, reference to “a compound” may refer to one or morecompounds, two or more compounds, three or more compounds, four or morecompounds, or greater numbers of compounds. The term “plurality” refersto “two or more.”

The use of the term “at least one” will be understood to include one aswell as any quantity more than one, including but not limited to, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” mayextend up to 100 or 1000 or more, depending on the term to which it isattached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y, and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y, and Z. The use of ordinal number terminology(i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for thepurpose of differentiating between two or more items and is not meant toimply any sequence or order or importance to one item over another orany order of addition, for example.

For purposes of the instant disclosure, the term “at least” followed bya number is used herein to denote the start of a range beginning withthat number (which may be a ranger having an upper limit or no upperlimit, depending on the variable being defined). For example, “at least1” means 1 or more than 1. The term “at most” followed by a number isused herein to denote the end of a range ending with that number (whichmay be a range having 1 or 0 as its lower limit, or a range having nolower limit, depending upon the variable being defined). For example,“at most 4” means 4 or less than 4, and “at most 40%” means 40% or lessthan 40%.

The use of the term “or” in the claims is used to mean an inclusive“and/or” unless explicitly indicated to refer to alternatives only orunless the alternatives are mutually exclusive. For example, a condition“A or B” is satisfied by any of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

As used herein, any reference to “one embodiment,” “an embodiment,”“some embodiments,” “one example,” “for example,” or “an example” meansthat a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. The appearance of the phrase “in some embodiments” or “oneexample” in various places in the specification is not necessarily allreferring to the same embodiment, for example. Further, all referencesto one or more embodiments or examples are to be construed asnon-limiting to the claims.

Similarly, it is to be understood that where the specification statesthat a component, feature, structure, or characteristic “may,” “might,”“can,” or “could” be included, that particular component, feature,structure, or characteristic, while present in one or more particular(but non-limiting) embodiments, is not required to be included in allembodiments.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for acomposition/apparatus/device, the method being employed to determine thevalue, or the variation that exists among the study subjects. Forexample, but not by way of limitation, when the term “about” isutilized, the designated value may vary by plus or minus twenty percent,or fifteen percent, or twelve percent, or eleven percent, or tenpercent, or nine percent, or eight percent, or seven percent, or sixpercent, or five percent, or four percent, or three percent, or twopercent, or one percent from the specified value, as such variations areappropriate to perform the disclosed methods and as understood bypersons having ordinary skill in the art.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”), or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, when associated with a particular event orcircumstance, the term “substantially” means that the subsequentlydescribed event or circumstance occurs at least 80% of the time, or atleast 85% of the time, or at least 90% of the time, or at least 95% ofthe time. For example, the term “substantially adjacent” may mean thattwo items are 100% adjacent to one another, or that the two items arewithin close proximity to one another but not 100% adjacent to oneanother, or that a portion of one of the two items is not 100% adjacentto the other item but is within close proximity to the other item.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, such as (but notlimited to) more than about 85%, 90%, 95%, and 99%. In a particular (butnon-limiting) embodiment, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods), wherein the composition consistsessentially of a single macromolecular species.

When, in this document, a range is given as “(a first number) to (asecond number)” or “(a first number)-(a second number),” this means arange whose lower limit is the first number and whose upper limit is thesecond number. For example, 25 to 100 should be interpreted to mean arange whose lower limit is 25 and whose upper limit is 100.Additionally, it should be noted that where a range is given, everypossible subrange or interval within that range is also specificallyintended unless the context indicates to the contrary. For example, ifthe specification indicates a range of 25 to 100, such range is alsointended to include subranges such as 26-100, 27-100, etc., 25-99,25-98, etc., as well as any other possible combination of lower andupper values within the stated range, e.g., 33-47, 60-97, 41-45, 28-96,etc. Note that integer range values have been used in this paragraph forpurposes of illustration only, and decimal and fractional values (e.g.,46.7-91.3) should also be understood to be intended as possible subrangeendpoints unless specifically excluded.

Where applicable, although state diagrams, flow diagrams or both may beused to describe embodiments, the present disclosure is not limited tothose diagrams or to the corresponding descriptions. For example, flowneed not move through each illustrated box or state, or in exactly thesame order as illustrated and described.

The term “method,” as used herein, may refer to manners, means,techniques, and procedures for accomplishing a given task including, butnot limited to, those manners, means, techniques, and procedures eitherknown to, or readily developed from known manners, means, techniques,and procedures by practitioners of the art to which the presentdisclosure belongs.

Methods of the present disclosure may be implemented by performing orcompleting manually, automatically, or a combination thereof, selectedsteps or tasks. In addition, unless otherwise indicated, the selectedsteps or tasks of each method may be performed simultaneously or whollyor partially sequentially. In addition, unless indicated otherwise, theorder and sequence of selected steps or tasks of each method are forpurposes of illustration only; changes may be made in the order andsequence of steps, so long as the method is capable of functioning inaccordance with the present disclosure. That is, where reference is madeherein to a method comprising two or more defined steps, the definedsteps can be carried out in any order or simultaneously (except wherecontext excludes that possibility). In addition, any of the methods ofthe present disclosure can also include one or more other steps whichare carried out before any of the defined steps, between two of thedefined steps, or after all of the defined steps (except where contextexcludes that possibility).

Turning now to the inventive concept(s), disclosed herein arebiocatalytic conversion systems and methods of producing and using same,particularly (but not limited to), in the production of at least onealcohol, at least one ketone, and/or at least one organic acid.

Certain non-limiting embodiments are directed to a biocatalyticconversion system that utilizes a co-fermentation process for sugar andgaseous substrates. The biocatalytic conversion system comprises tworeactors with a gas line connecting the two. The first reactor comprisesat least one fermentation medium containing at least one firstmicroorganism. The at least one fermentation medium comprises at leastone sugar substrate, and the at least one first microorganism comprisesa sugar fermenting species that converts sugars into at least one ofacetone, butanol, ethanol, isopropanol, acetic acid, and butyric acid.In addition, a gaseous substrate comprising CO₂ and H₂ gases is producedduring the fermentation process. The second reactor comprises at leastone medium containing at least one second microorganism, wherein the atleast one second microorganism comprises a gas fermenting species thatconverts CO₂ and H₂ gases into at least one of an alcohol and an organicacid. The gas line connects the first reactor to the second reactor forfeeding the gaseous substrate produced in the first reactor into thesecond reactor. For example (but not by way of limitation), the gas linemay connect a headspace of the first reactor to a headspace of thesecond reactor. Alternatively, gas transferred from the first reactorcan be bubbled into the liquid medium of the second reactor. Inaddition, the biocatalytic conversion system is absent a second line forfeeding a substrate produced in the second reactor into the firstreactor.

In certain particular (but non-limiting) embodiments, the volume of thesecond reactor is larger than the volume of the first reactor. The ratioof first reactor volume to second reactor volume is selected to allowfor the flow of gas from the first reactor to the second reactor withoutover pressurizing second reactor, which would hinder gas flow from thefirst reactor to the second reactor. When the pressure in the secondreactor is higher than the pressure in the first reactor, the firstreactor will cease producing H₂ and CO₂. For example (but not by way oflimitation), the ratio of first reactor volume to second reactor volumemay be about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7,about 1:8, about 1:9, about 1:10, or larger, as well as within a rangeformed of any of the above values (i.e., a range of from about 1:2 toabout 1:10, etc.).

Certain non-limiting embodiments of the present disclosure are alsodirected to a biocatalytic conversion system that utilizes aco-fermentation process for sugars and gases. These embodiments aresimilar to the biocatalytic conversion system above except that allcomponents are placed within a single reactor. That is, the biocatalyticconversion system comprises a reactor that includes at least onefermentation medium and two species of microorganisms. The at least onefermentation medium comprises at least one sugar substrate. The at leastone first microorganism comprises a sugar fermenting species thatconverts sugars into at least one of acetone, butanol, ethanol,isopropanol, acetic acid, and butyric acid, and wherein a gaseoussubstrate comprising CO₂ and H₂ gases is produced during thefermentation process. The at least one second microorganism comprises agas fermenting species that converts CO₂ and H₂ gases produced duringthe fermentation of sugars into at least one of an alcohol and anorganic acid.

In a particular (but non-limiting) embodiment, the biocatalyticconversion systems disclosed or otherwise contemplated herein produce atleast one alcohol, at least one ketone, and at least one organic acid.

Certain non-limiting embodiments of the present disclosure are directedto a method of biocatalytic conversion that utilizes a co-fermentationprocess for sugar and gaseous substrates. The method comprises the stepsof: (a) contacting at least one fermentation medium with at least onefirst microorganism in a first reactor, wherein the at least onefermentation medium comprises at least one sugar substrate, and whereinthe at least one first microorganism converts the at least one sugarsubstrate into at least one of acetone, butanol, ethanol, isopropanol,acetic acid, and butyric acid, and wherein a gaseous substratecomprising CO₂ and H₂ gases is produced during the fermentation process;and (b) feeding the gaseous substrate produced in the first reactor intoa second reactor, the second reactor comprising at least one mediumcontaining at least one second microorganism, wherein the at least onesecond microorganism converts CO₂ and H₂ gases produced in the firstreactor and fed into the second reactor into at least one of an alcoholand an organic acid.

In certain particular (but non-limiting) embodiments of the abovemethod, the method further includes the steps of preparing an inoculumpreparation of the at least one second microorganism by pre-culturingthe at least one second microorganism in the presence of a gas mixturecomprising at least three gases selected from the group consisting ofcarbon monoxide, carbon dioxide, hydrogen, and nitrogen, and theninoculating the second reactor with the inoculum preparation of the atleast one second microorganism. Any gas mixtures disclosed herein orotherwise known in the art that can function to prepare the secondmicroorganism to function in accordance with the present disclosure maybe utilized in accordance with the scope of the present disclosure. Forexample (but not by way of limitation), the gas mixture may comprise atleast three of: carbon monoxide gas in a range of from about 20% toabout 40% by volume; carbon dioxide gas in a range of from about 15% toabout 30% by volume; hydrogen gas in a range of from about 5% to about60% by volume; and nitrogen gas in a range of from about 20% to about60% by volume. In a particular (but non-limiting) example, the gasmixture comprises about 40% CO, about 30% CO₂, and about 30% H₂ byvolume. In another particular (but non-limiting) example, the gasmixture comprises about 20% CO₂, about 60% H₂, and about 20% N₂, byvolume.

Certain non-limiting embodiments of the present disclosure are directedto a method of biocatalytic conversion that utilizes a co-fermentationprocess for sugar and gaseous substrates. The method comprising thesteps of: (a) contacting at least one fermentation medium with at leastone first microorganism in a reactor, wherein the at least onefermentation medium comprises at least one sugar substrate, and whereinthe at least one first microorganism converts the at least one sugarsubstrate into at least one of acetone, butanol, ethanol, isopropanol,acetic acid, and butyric acid, and wherein a gaseous substratecomprising CO₂ and H₂ gases is produced during the fermentation process;and (b) contacting the gaseous substrate produced during thefermentation process with at least one second microorganism present inthe reactor, wherein the at least one second microorganism converts CO₂and H₂ gases produced during the fermentation of sugars into at leastone of an alcohol and an organic acid.

In certain particular (but non-limiting) embodiments of the abovemethod, the method further includes the steps of preparing an inoculumpreparation of the at least one second microorganism by pre-culturingthe at least one second microorganism in the presence of a gas mixturecomprising at least three gases selected from the group consisting ofcarbon monoxide, carbon dioxide, hydrogen, and nitrogen, and theninoculating the reactor with the inoculum preparation of the at leastone second microorganism. Any gas mixtures disclosed herein or otherwiseknown in the art that can function to prepare the second microorganismto function in accordance with the present disclosure may be utilized inaccordance with the scope of the present disclosure. For example (butnot by way of limitation), the gas mixture may comprise at least threeof: carbon monoxide gas in a range of from about 20% to about 40% byvolume; carbon dioxide gas in a range of from about 15% to about 30% byvolume; hydrogen gas in a range of from about 5% to about 60% by volume;and nitrogen gas in a range of from about 20% to about 60% by volume. Ina particular (but non-limiting) example, the gas mixture comprises about40% CO, about 30% CO₂, and about 30% H₂ by volume. In another particular(but non-limiting) example, the gas mixture comprises about 20% CO₂,about 60% H₂, and about 20% N₂, by volume.

Any biocatalytic species (or combinations thereof) that are known in theart or otherwise contemplated herein and that can function in accordancewith the present disclosure are included within the scope of the systemsand methods described herein. That is, any type of saccharolyticspecies, or any co-culture or mixed culture containing one or moresaccharolytic species, may be utilized as the at least one firstmicroorganism. Likewise, any microbial catalyst capable of fermentingsynthesis gas (“syngas,” which typically comprises CO, CO₂, H₂, and/orN₂) to produce one or more liquid biofuels or chemicals, as well as anyco-culture or mixed culture containing one or more of said microbialcatalysts, may be utilized as the at least one second microorganism.

In certain particular (but non-limiting) embodiments, each of the firstand second microorganisms comprises one or more individual species ofmicroorganisms, wherein each individual species is from a genus selectedfrom the group consisting of Clostridium, Butyribacterium, Eubacterium,Moorella, Acetobacterium, Enterobacter, Bacillus, Anaerobaculum,Alkalibaculum, and combinations thereof. For example, but not by way oflimitation, the at least one first microorganism may comprise at leastone of Clostridium acetobutylicum, Bacillus firmus, Anaerobaculumhydrogeniformans, and Clostridium beijerinckii. In another non-limitingexample, the at least one second microorganism may comprise at least oneof Clostridium ragsdalei, Clostridium autoethanogenum, Clostridiumcarboxidivorans, Clostridium ljungdahlii, Clostridium muellerianum, andAlkalibaculum bacchi.

In a particular (but non-limiting) embodiment, each of the first andsecond microorganisms comprises a Clostridium species, and each of thefirst and second microorganisms may contain a mono-culture or aco-culture/mixed culture containing said Clostridium species. Aparticular (but non-limiting) example of bacteria that may be utilizedas the at least one first microorganism comprises Clostridiumacetobutylicum ATCC 824 (including a co-culture or mixed culturecontaining same). A particular (but non-limiting) example of bacteriathat may be utilized as the at least one second microorganism comprisesClostridium ragsdalei P11 (including a co-culture or mixed culturecontaining same).

When the at least one second microorganism comprises Clostridiumragsdalei (including a co-culture or mixed culture containing same),ethanol, acetic acid, and isopropanol can be produced in the reactorcontaining the at least one second microorganism. When the at least onesecond microorganism comprises Clostridium carboxidivorans (including aco-culture or mixed culture containing same), ethanol, butanol, hexanol,acetic acid, butyric acid, and hexanoic acid can be produced in thereactor containing the at least one second microorganism. When the atleast one second microorganism comprises Clostridium muellerianum(including a co-culture or mixed culture containing same), ethanol,butanol, hexanol, acetic acid, butyric acid, and hexanoic acid can beproduced in the reactor containing the at least one secondmicroorganism.

Any fermentation media that can support the functions of the firstand/or second microorganisms can be utilized in accordance with thesystems and methods of the present disclosure. In certain non-limitingembodiments, the sugar substrate present in the fermentation medium isselected from the group consisting of glucose, fructose, sucrose,xylose, galactose, arabinose, mannose, and combinations thereof.However, other sugar substrates that can be broken down bymicroorganisms can also be utilized in accordance with the presentdisclosure.

In certain non-limiting embodiments, the fermentation medium contains apure (or substantially pure) sugar for use in the production ofalcohol(s), ketone(s), and/or organic acid(s). Alternatively, thefermentation may contain any type of material that can comprise one ormore sugars for use in the production of alcohol(s), ketone(s), and/ororganic acid(s). In certain non-limiting embodiments, the fermentationmedium comprises at least one raw material selected from the groupconsisting of a sugar, a starch, cellulose, hemicellulose, othercarbohydrates, glucan, xylan, galactan, mannan, cellobiose, othercarbohydrates, lignocellulosic biomass (such as, but not limited to,grasses and wood materials), wastes containing lignocellulosicmaterials, and combinations thereof. In certain non-limitingembodiments, the fermentation medium contains a feedstock selected fromthe group consisting of switchgrass, forage sorghum, redcedar, woodymaterials, and combinations thereof. When a feedstock is present in thefermentation medium, the feedstock may be pretreated and hydrolyzedprior to placement in the fermentation medium, so as to release sugarsfor fermentation.

The medium fed into the reactor containing the at least one firstmicroorganism may contain any concentration of sugar and/or feedstock,so long as the biocatalytic conversion system is capable of functioningas described herein to produce one or more products (including, but notlimited to, one or more of acetone, butanol, ethanol, isopropanol,acetic acid, butyric acid, as well as one or more other alcohols and/ororganic acids) with any level of yield. For example (but not by way oflimitation), the feedstock may be present in the medium in aconcentration of at least about 0.01%, at least about 0.05%, at leastabout 0.1%, at least about 0.5%, at least about 1%, at least about 2%,at least about 3%, at least about 4%, at least about 5%, at least about6%, at least about 7%, at least about 8%, at least about 9%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 76%, at least about 77%, at least about 78%, at least about 79%,at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, and at least about 99%. In addition, thescope of the presently disclosure also includes the presence offeedstock in the medium at any percent yield that falls within any rangeformed from the combination of two values listed above (for example, arange of from about 10% to about 99%, a range of from about 30% to about98%, a range of from about 50% to about 97%, a range of from about 60%to about 96%, a range of from about 70% to about 95%, etc.).

The reactors utilized in the biocatalytic conversion systems disclosedherein may be maintained at any temperature and any pH that allows thereactors to perform as described or otherwise contemplated herein. In aparticular (but non-limiting) embodiment, when two reactors are presentin the biocatalytic conversion system, the first reactor is maintainedat a temperature in a range of from about 20° C. to about 45° C., suchas (but not limited to) about 37° C., while a pH of the at least onefermentation medium present in the first reactor is maintained in arange of from about 4.0 to about 7.5, such as (but not limited to)between about 6.5 and about 6.8. Also in this particular (butnon-limiting) embodiment, the second reactor is maintained at atemperature in a range of from about 20° C. to about 45° C., such as(but not limited to) about 37° C., while a pH of the at least onefermentation medium present in the second reactor is maintained in arange of from about 4.0 to about 7.5, such as (but not limited to) about6.0. The use of thermophilic microorganisms in the biocatalyticconversion system requires a higher temperature range of from about 20°C. to about 65° C., such as (but not limited to) about 55° C. In anotherparticular (but non-limiting) embodiment, when a single reactor ispresent in the biocatalytic conversion system, the one reactor ismaintained at a temperature in a range of from about 20° C. to about 45°C., such as (but not limited to) about 37° C., while a pH of the atleast one fermentation medium present in the reactor is maintained in arange of from about 4.0 to about 7.5, such as (but not limited to) about6.5.

In certain particular (but non-limiting) embodiments, the biocatalyticconversion systems and methods of the present disclosure may furtherinclude the step of filling the headspace of the reactor containing theat least one first organism (i.e., the first reactor or single reactor)with a CO₂-containing gas prior to inoculation with the at least onefirst organism. For example, but not by way of limitation, the headspaceof the first organism-containing reactor may be filled with a gascomprising a CO₂ concentration of about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or about 100%, as well as a CO₂ concentrationfalling within a range of two of the above values (i.e., a range of fromabout 40% to about 100%, a range of from about 40% to about 60%, etc.).

In certain particular (but non-limiting) embodiments, the biocatalyticconversion systems and methods of the present disclosure may furtherinclude the addition of an external gas feed into the reactor containingthe at least one second microorganism. For example (but not by way oflimitation), external CO and/or H₂ gas may be fed into the singlereactor system or into the second reactor of a two-reactor system.

In certain particular (but non-limiting) embodiments, the reactorcontaining the at least one second microorganism (i.e., the secondreactor or single reactor) has a ratio of CO₂ to H₂ of about 1:4, about1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or higher,as well as a range formed of two of the above values (i.e., a range offrom about 1:4 to about 4:1).

Each of the systems and methods disclosed or otherwise contemplatedherein may be performed in batch reactions or via fed-batch orcontinuous flow methodologies. That is, the systems and methods mayinclude the use of batch reactors or continuous flow reactors.

Each of the systems or methods described or otherwise contemplatedherein can produce one or more products (including, but not limited to,one or more of acetone, butanol, ethanol, isopropanol, acetic acid,butyric acid, as well as one or more other alcohols and/or organicacids) with any level of yield. For example (but not by way oflimitation), the one or more products can be produced with a yield of atleast about 0.01%, at least about 0.05%, at least about 0.1%, at leastabout 0.5%, at least about 1%, at least about 2%, at least about 3%, atleast about 4%, at least about 5%, at least about 6%, at least about 7%,at least about 8%, at least about 9%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, and at least about 99%. In addition, the scope of the presentlydisclosure also includes the production of one or more products at anypercent yield that falls within any range formed from the combination oftwo values listed above (for example, a range of from about 10% to about99%, a range of from about 30% to about 98%, a range of from about 50%to about 97%, a range of from about 60% to about 96%, a range of fromabout 70% to about 95%, etc.).

In particular (but not by way of limitation), there is provided hereinan embodiment of an integrated conversion process that uses a novelco-fermentation process (see FIG. 1 ). One non-limiting objective of thepresent disclosure is to establish an integrated novel conversionprocess for production of butanol and other alcohols utilizing novelbiocatalytic conversion processes. Various embodiments can be performedin one reactor in a co-culture/mixed culture, or in two reactors asseparate cultures (including two separate mono-cultures, co-cultures,and/or mixed cultures) to produce various products (FIG. 1 ). Variousfeedstocks containing sugars, starch, cellulose, hemicellulose, and/orother carbohydrates can be used in the present disclosure. Some of thefeedstocks should be processed first to release the sugars to be used bythe biocatalyst in the present disclosure. Existing methods to releasesugars from starch and lignocellulosic feedstocks can be used; as thesemethods are well known in the art, no further description thereof isdeemed necessary.

According to one particular (but non-limiting) embodiment, there isprovided a process that consists of a co-fermentation of sugars andgaseous substrates for ABE production in a single fermentation systemwhile converting CO₂ and H₂ to alcohols and organic acids, thus addingextra revenue to biorefineries. The raw materials can be starch andsugar crops or lignocellulosic biomass. Depending on the microorganismsand reactor configuration used, an embodiment can enhance alcohol yieldby more than 25%. In one example, the use of two-stage reactors resultedin a 19% improvement in ABE yield from sugar conversion by C.acetobutylicum ATCC 824 in the first stage and conversion of H₂ and CO₂by C. ragsdalei in the second reactor. The total organic acid productionin the co-fermentation process in the two-stage reactors was alsoincreased by 141% due to conversion of H₂ and CO₂. In addition toreducing CO₂ emissions, the co-fermentation of sugars and gases has thepotential to enhance the feasibility of ABE fermentation by producingmore alcohols from generated waste gas streams.

Various feedstocks containing sugars, starch, cellulose, hemicellulose,and/or other carbohydrates can be used in the present disclosure. Someof the feedstocks may need to be processed first to release the sugarsto be used by the biocatalyst in the present disclosure. Existingmethods to release sugars from starch and lignocellulosic feedstocks canbe used; as these methods are well known in the art, no furtherdescription thereof is deemed necessary.

Examples of feedstocks that can be used include (but are not limited to)switchgrass, forage sorghum, redcedar, and woody materials. Pretreatedswitchgrass and redcedar will be hydrolyzed using commercial enzymes torelease the sugars for the fermentation process. After hydrolysis ofpretreated biomass, the fermentation process can proceed in two routesaccording to the embodiments shown in FIG. 1 . In Route 1 (two stagereactor), the released sugars are fermented in the first reactor usingat least one first microorganism (such as, but not limited to, amono-culture of Clostridium acetobutylicum) to acetone, butanol, andethanol (ABE), and the generated H₂ and CO₂ are fermented in the secondreactor by a at least one second microorganism (such as, but not limitedto, Clostridium ragsdalei or a co-culture or mixed culture ofmicroorganisms containing same) to ethanol and acetic acid. In Route 2(single reactor), the released sugars and generated H₂ and CO₂ areconverted to isopropanol, butanol, and ethanol (IBE) as well as organicacids using a co-culture or mixed culture of first and secondmicroorganisms (such as, but not limited to, a co-culture comprising C.acetobutylicum and C. ragsdalei). Butanol is the main product in thisprocess from both routes. However, the process route, microorganisms,and operating conditions utilized affect the product ratios and yields.Separate hydrolysis and fermentation, simultaneous saccharification andco-fermentation, and other fermentation schemes can be used. C.acetobutylicum and C. ragsdalei require similar growth medium andconditions for growth such as temperature and pH. C. acetobutylicumconverts C5 and C6 sugars to ABE, in a typical product ratio of about3:6:1. C. ragsdalei, developed through collaborative efforts between OSUand OU (U.S. Pat. No. 7,704,723, issued to Huhnke et al. on Apr. 27,2010, the entire contents of which are hereby expressly incorporatedherein by reference), converts acetone into isopropanol, organic acidsinto alcohols, and H₂ and CO₂ into ethanol and acetic acid. Also, whilecertain particular species have been described herein, it will beunderstood that the systems and methods of the present disclosure can beused with any other combination of microorganisms that are capable ofconverting sugars and CO/CO₂/H₂ into various products to support food,agriculture, pharmaceutical, environmental, and energy industries.

The present disclosure is the first report to describe theco-fermentation of sugars and gases for alcohol and organic acidproduction using pure sugars or sugars derived from biomass.

The foregoing has outlined in broad terms some of the more importantfeatures of the present disclosure so that the detailed description thatfollows may be more clearly understood, and so that the contribution ofthe instant inventors to the art may be better appreciated. Theinventive concept(s) is not to be limited in its application to thedetails of the construction and to the arrangements of the componentsset forth in the following description or illustrated in the drawings.Rather, the inventive concept(s) is capable of other embodiments and ofbeing practiced and carried out in various other ways not specificallyenumerated herein. Finally, it should be understood that the phraseologyand terminology employed herein are for the purpose of description andshould not be regarded as limiting, unless the specificationspecifically so limits the inventive concept(s).

EXAMPLE

Examples are provided hereinbelow. However, the present disclosure is tobe understood to not be limited in its application to the specificexperimentation, results, and laboratory procedures disclosed herein.Rather, the Examples are simply provided as one of various embodimentsand are meant to be exemplary, not exhaustive.

To address the defects and disadvantages of the prior art discussed indetail in the Background section, an integrated conversion process usinga novel co-fermentation process has been developed (FIG. 1 ). Variousfeedstocks containing sugars, starch, cellulose, hemicellulose, and/orother carbohydrates can be used with the novel co-fermentation process.Some feedstocks should be pretreated and hydrolyzed to release thesugars for fermentation. In addition, separate hydrolysis andfermentation, simultaneous saccharification and co-fermentation, andother fermentation schemes can be used with this process.

In Route 1 of FIG. 1 , two reactors are used in series to make ABEproducts. In the first reactor, sugar fermenting microorganisms such asClostridium acetobutylicum (Ca) or Clostridium beijerinckii (Cb) areused to convert sugars into ABE as well as acetic and butyric acids. TheCO₂ and H₂ produced in the first reactor are fed to the second reactorcontaining gas fermenting microorganisms such as Clostridium ragsdalei(Cr) or Clostridium carboxidivorans (Cc) to make additional alcohols andorganic acids. Route 2 shows an example of a co-culture of sugarfermenting microorganism (Ca) and gas fermenting microorganism (Cr) usedin one reactor. Route 2 is more desirable than route 1 because it savesmore capital and operating costs using one reactor. In this novelprocess, more ethanol and acetic acid are produced when Cr is used.Also, when Cr is used in Route 2, acetone is converted to isopropanol.However, when Cc is used in Route 2, more butanol and butyric acid areproduced. Unlike Cr, Cc does not have the ability to convert acetone toisopropanol (Ramachandriya et al., 2011).

The methods of the present disclosure can also be used with othermicroorganisms to produce various products benefiting from theco-utilization of sugars and gases. Butanol is the main product in thisprocess from both routes. However, the process route, microorganisms,and operating conditions utilized affect the product ratios and yields.When feasible, the addition of external CO or H₂ into the reactor withthe gas fermenting microorganism can increase alcohol formation andfurther reduces CO₂ emission.

The current Examples focus on co-fermentation of sugars and gaseoussubstrates for ABE and organic acid production in a single fermentationsystem. Therefore, this approach of co-fermentation enhances the processeconomy by generating extra income utilizing the off-gas streams whilealso reducing CO₂ emissions. Pure sugars and sugars derived from woodybiomass (such as, but not limited to, eastern redcedar) were used in theExamples. In particular, in Example 2, the eastern redcedar was used asa feedstock for the co-fermentation process following pretreatment,enzymatic hydrolysis, and detoxification thereof.

The present disclosure is the first report of co-fermentation of sugarsand gases for production of alcohol and organic acids using pure sugarsor sugars derived from biomass. For example (but not by way oflimitation), the use of C. acetobutylicum ATCC 824 and C. ragsdalei intwo-stage reactors has shown a 19% improvement in ABE(acetone-butanol-ethanol) yield due to additional ethanol produced fromH₂ and CO₂. In addition, total organic acid production in theco-fermentation process increased by 141% due to utilization of H₂ andCO₂ (FIGS. 3 and 4 ). In addition to reducing CO₂ emissions, the novelco-fermentation of sugars and gases enhances the process economics byproducing more products from wasted gas streams.

Example 1—Co-Fermentation of Pure Glucose and Generated CO₂ and H₂Methods of Example 1

Microorganisms and Inoculum Preparation

Clostridium ragsdalei strain P11 and Clostridium acetobutylicum ATCC 824were used in the study. C. acetobutylicum culture was maintained asspore suspension in sterile distilled water at 4° C. (Liu et al.,2015b). Tryptone-glucose-yeast (TGY) medium was used in pre-culturing ofC. acetobutylicum. The TGY medium contained (per liter): 30 g tryptone,20 g glucose, 10 g yeast extract, and 1 g of cysteine (Ezeji et al.,2013). The TGY medium was autoclaved for 15 min at 121° C. (PRIMUS,Sterilizer CO. Inc., Omaha, Nebr., USA) with the reactor bottles, tips,and test tubes before the inoculation. The heat shock protocol of C.acetobutylicum and inoculation procedures were previously described (Liuet al., 2015b). C. ragsdalei was pre-cultured on standard medium thatcontained (per liter): 1 g yeast extract, 25 mL mineral stock solution,10 mL vitamin stock solution, 10 mL trace metals stock solution, 10 gN-morpholinoethane-sulfonic acid (MES) buffer, 2.5 mL of 4% cysteinesulfide solution, and 1 mL of 0.1% resazurin solution. The mineral stocksolution contained (per liter): 100 g ammonium chloride, 20 g magnesiumsulfate, 10 g potassium chloride, 10 g potassium phosphate monobasic,and 4 g calcium chloride. The vitamin stock solution contained (perliter): 10 mg MESNA (2-mercaptoethanesulfonic acid sodium salt), 10 mgpyridoxine, 5 mg p-(4)-aminobenzoic acid, 5 mg calcium pantothenate, 5mg nicotinic acid, 5 mg riboflavin, 5 mg thiamine, 5 mg thioctic acid, 5mg vitamin B12, 2 mg d-biotin, and 2 mg folic acid. The trace metalstock solution contained (per liter): 2.0 g nitrilotriacetic acid, 1.0 gmanganese sulfate, 1.0 g zinc sulfate, 0.8 g ferrous ammonium sulfate,0.2 g cobalt chloride, 0.2 g nickel chloride, 0.2 g sodium tungstate,0.1 g sodium selenate, and 0.02 g sodium molybdate. The pH of C.ragsdalei medium was adjusted during preparation to 6.0 using 5N KOHsolution.

Pre-culturing of C. ragsdalei in Passage I was performed in serumbottles containing standard C. ragsdalei medium (45 mL working volume)and 5 mL of seed culture. Then, the culture bottles were flushed for 3min with synthesis gas (syngas) Mix I, which contained 20% CO, 15% CO₂,5% H₂, and 60% N₂, by volume. The bottles were pressurized to 170.2 kPa(abs) with syngas Mix I and stored in a warm room (37° C.) withoutagitation. After two days, the bottles were placed on a shaker at 150rpm and 37° C. Once the optical density (OD) of culture in Passage Ireached 0.5 units, the culture from Passage I was used to inoculatefresh medium in Passage II for further adaptation to higher levels ofCO, CO₂, and H₂. The gas composition used in Passage II was syngas MixII containing 40% CO, 30% CO₂, and 30% H₂, by volume. Passage II bottleswere flushed with syngas Mix II for 3 min prior to inoculation of C.ragsdalei from Passage I. After inoculation, Passage II bottles werepressurized to 239.1 kPa (abs) with syngas Mix II and placed on a shakerat 150 rpm and 37° C. Once the OD in Passage II bottles reached about 1OD unit (usually within 24 to 36 h), this culture was used to inoculatethe gas fermentor (Fermentor B) in FIG. 2 .

Reactor Configuration

The co-fermentation system consisted of a 250 mL (Fermentor A for C.acetobutylicum) and a 1-L (Fermentor B for C. ragsdalei) Corning glassbottles with rubber stoppers to maintain the gastight operation (FIG. 2). The headspaces of Fermentors A and B were connected with ⅛″ innerdiameter stainless steel tubing. Similar diameter tubing with two valveswas used to obtain liquid samples from both fermentors. The headspace inthe two fermenters before inoculation contained 10% CO, 52.2% CO₂, and37.8% H₂, by volume, at 101.3 kPa (abs). After inoculation, Fermentor Bwas kept on a magnetic stirrer, and the stirring speed was maintained at150 rpm. The initial working volume in each of Fermentor A and FermentorB was 50 mL.

Fermentation Medium

ABE fermentations were performed in Fermentor A using two mediumformulations.

Medium I (used in Example 1) was a pure sugar medium, and Medium II(used in Example 2) contained detoxified redcedar hydrolyzate. Medium Iconsists of P2 medium and 60 g/L glucose and about 2 g/L xylose. MediumII contained P2 medium and redcedar hydrolyzate with similar glucose andxylose concentrations as in Medium I. Pre-culturing of C. acetobutylicum(ATCC 824) was carried out with TGY medium as discussed in the“Microorganisms and inoculum preparation” section. Medium I wasautoclaved at 121° C. for 15 min and cooled down to 40° C. prior totransfer into the anaerobic chamber. Medium I was then transferred intopre-sterile loosely capped 250 mL Fermentor A and supplemented with 2%(v/v) of pre-sterile yeast extract (50 g/L), 5% (v/v) of 1M acetatebuffer solution at pH 5.5, and 1% (v/v) of P2 vitamin, buffer, and tracemetal solutions. Prior to inoculation, the pH of Medium I was adjustedto between 6.5 and 6.8 using 2N KOH. Medium I was then inoculated with6% (v/v) actively growing C. acetobutylicum pre-cultured in TGY medium.Medium II contained P2 vitamin, buffer, and trace metal solutions, andredcedar hydrolyzate. The redcedar hydrolyzate was diluted to achieve aglucose concentration of 60 g/L in Medium II. No pH adjustment wasrequired during Medium II preparation.

Conversion of CO₂ and H₂ occurred in Fermentor B. Standard C. ragsdaleimedium was used as described in the “Microorganisms and inoculumpreparation” section. Prior to inoculation, about 60% (v/v) of freshsterile C. ragsdalei medium was added into Fermentor B. A 4% cysteinesulfide solution at 0.25% (v/v) was then added to deoxygenate and reducethe medium. Then, 40% (v/v) of actively growing C. ragsdalei pre-culturefrom Passage II was transferred aseptically into Fermentor B at 6 hoursafter starting ABE fermentation in Fermentor A. Both Fermentors A and Bwere maintained at 37° C.

Analytical Methods

During fermentation, 1 mL liquid samples from Fermentors A and B wereaseptically taken at various times to determine cell growth, pH, andproduct profiles. The optical density (OD) of the fermentation mediumfrom the liquid samples from Fermentors A and B were determined using aUV spectrophotometer (UV-2100) at 600 nm and 660 nm, respectively. Sugarconcentration in Fermentor A was analyzed using a HPLC (HPLC1200,Agilent Technologies, Wilmington, Del., USA). However, solvents,ketones, and organic acids produced in Fermentors A and B were measuredusing GC-FID (GC 6890, Agilent Technologies, Wilmington, Del., USA) aspreviously described (Liu et al., 2012; and Ramachandriya et al., 2013).The gas samples from headspaces of each fermentor were analyzed usingGC-TCD (Liu et al., 2012).

Results and Discussion of Example 1

Co-Fermentation of Pure Glucose and Generated CO₂ and H₂

Results of co-fermentation of pure sugars in Medium I by C.acetobutylicum (ATCC 824) and the generated CO₂ and H₂ by C. ragsdaleiare shown in FIGS. 3 and 4 . The OD of C. acetobutylicum cultureincreased to about 7.5 units, while the pH dropped from 6.6 to 5.6 (FIG.3 , Panel A). The pH of Medium I in Fermentor A was not adjusted. After72 h of fermentation, 48.5 g/L of glucose was consumed by C.acetobutylicum (FIG. 3 , Panel B). Fermentation results showed that 9.7g/L of butanol, 13.9 g/L of total ABE, and 3.3 g/L of total acids wereproduced (FIG. 3 , Panels C and D).

On the other hand, C. ragsdalei grew in Fermentor B on H₂ and CO₂ andreached a maximum OD of 0.8 while producing 2.6 g/L of ethanol from theoff gases (H₂ and CO₂) produced from Fermentor A (FIG. 4 , Panels A andC). Fermentor B produced about 19% additional solvent compared to totalABE produced in Fermentor A. Further, the total acids production inFermentor B was 4.7 g/L. Total organic acid concentration in bothFermentors increased by 141% due to H₂ and CO₂ utilization by C.ragsdalei. Therefore, the total ABE and organic acids produced from bothFermentors A and B were 16.5 g/L and 8.0 g/L, respectively.

FIG. 4 shows the cumulative production/consumption of different gascomponents (H₂, CO₂, and CO). The H₂ and CO₂ conversion efficienciesduring pure glucose fermentation were 72% and 16%, respectively.Further, the CO₂ accumulation in the co-fermentation system confirms theproduction of CO₂ is out competed by its consumption due to insufficientavailability of reductant gases generated during ABE fermentation. Theaddition of more H₂ or CO in Fermentor B is expected to increase the CO₂conversion to ethanol and acetic acid.

Example 2—Co-Fermentation of Detoxified Redcedar Hydrolyzate andGenerated CO₂ and H₂ Methods of Example 2

The methods of Example 2 were performed as described above in Example 1,with the additional methods described herein below.

Redcedar Pretreatments

Redcedar biomass pretreatment was performed in a 1.0 L Parr reactor(Parr series 4525, Parr Instrument Company, Moline, Ill.). The reactorwas equipped with a pressure gauge, heater, an agitator, and acontrolling module. According to the particle size distribution (Table1), 98.5% of the particles were 2.0 mm. The amount of redcedar biomassin each pretreatment experiment was determined based on the initialmoisture content of the biomass samples. During pretreatment, a 100.0 gof dry redcedar biomass was impregnated at 90° C. for 3.0 h inpretreatment liquor consisting of 3.75 g of sulfuric acid and 20.0 g ofsodium bisulfite. In this case, the ratio of pretreatment liquor tobiomass was maintained at 5:1 (v/w). Immediately after the 3.0 h ofimpregnation, the temperature of the reactor was increased to 200° C.and maintained for 10 min (Ramachandriya et al., 2013). The internalpressure of the reactor was recorded separately to confirm therepeatability of the pretreatment process. Immediately after 10.0 min oftemperature holding time, the reactor was immersed in an ice bath withmanually agitation of the pretreated biomass liquor until thetemperature of the reactor dropped to 55° C. After cooling the reactorbelow 55° C., the solids were separated by vacuum filtration.Subsequently, the filtered wet solids were washed and filtered fourtimes using 500 mL of preheated-deionized water at 60° C. The washed,pretreated redcedar biomass was then stored at 4° C. for further use.The composition of pretreated redcedar was measured as per the NationalRenewable Energy Laboratory (NREL) protocols (Sluiter et al., 2008).

Enzymatic Hydrolysis

Pretreated redcedar solids were subjected to enzymatic hydrolysis tofurther breakdown the biomass structure to obtain sugars. ACCELLERASE®1500 cellulase enzyme complex (Genencor Inc., Palo Alto, Calif., USA)was used. Experiments were performed in 250 mL Erlenmeyer flasks with atotal weight of 100 g (including pretreated biomass, water, and enzyme)in each flask and with shaking in an incubator shaker (MaxQ 4450,Thermos Scientific, Dubuque, Iowa, USA) at 250 rpm. A solid loading of14% was selected in order to obtain 60 g/L glucose for ABE fermentation.The enzyme loading was 50 FPU/g of glucan as previously used (Liu etal., 2015b). A sample of 2.0 mL was taken aseptically from each flask at6, 12, 24, and 48 h to measure sugar yield.

Detoxification of Enzymatic Hydrolysis

Once the enzymatic hydrolysis was completed (after 48 h), the hydrolyzedredcedar slurry was centrifuged (Avanti J-E, Beckman Coulter, Inc.,Brea, Calif., USA) for 15 min at 48,000 g and 4° C. to separate thesolids. The centrifugation was continued four times to separatesuspended redcedar solids from liquid hydrolyzate before detoxification.The soluble lignin content (SLC) was measured as previously described byMussatto and Roberto (2006), and bisulfite content was measured aspreviously described by Liu et al. (2015b) before and after thedetoxification of hydrolyzate. Detoxification was performed to removephenolic inhibitors from the hydrolyzate with 10% (w/v) powderedactivated carbon (Hydrodarco B, CABOT, Norit American Inc., Marshall,Tex., USA) (Liu et al., 2015b). The activated carbon was mixed with thehydrolyzate at 250 rpm and 28° C. for 1 h. The activated carbon was thenremoved from the hydrolyzate by centrifugation at 48,000 g and 4° C. for15 min. After centrifugation, the hydrolyzate was filter-sterilizedusing 0.2 μm nylon filters (NALGENE® RAPID-FLOW® filter units,ThermoFisher, Waltham, Mass., USA) and stored in −20° C. freezer forfurther use.

Results and Discussion of Example 2

Pretreatment of Redcedar Biomass

The redcedar used in the present Example has various particle sizes;thus, a particle size distribution analysis was performed prior to thepretreatment (Table 1). About 1.5% of the total redcedar particles werelarger than 2.0 mm. However, 11.3% of the particles were retained in0.25 mm sieve opening. Nearly 40% of the particles were retained in 1.0mm sieve. Over 85% of redcedar particle size was between 0.25 mm and 2.0mm.

Acid-bisulfite pretreatment plays an important role of breaking down theredcedar biomass structure and exposing cellulose and hemicellulose forenzymatic hydrolysis. After the acid-bisulfite pretreatment, sevenpretreated redcedar biomass samples were mixed before enzymatichydrolysis. According to the compositional analysis, the mixedpretreated redcedar biomass contained 53.53±0.34% of glucan, 2.55±0.04%of xylan, 1.33±0.03% of galactan, 2.38±0.16% of mannan, and 32.91±1.14%of lignin.

TABLE 1 Particle size distribution of the redcedar used in the studySieve size Mass percent retained (mm) on screen (%) >2.0  1.5 ± 0.1 2.016.3 ± 1.3 1.0 38.4 ± 1.7 0.85  8.7 ± 0.1 0.6 10.6 ± 0.3 0.355  8.7 ±0.4 0.25  4.5 ± 0.7 <0.25 11.3 ± 1.6

Enzymatic Hydrolysis and Detoxification

Enzymatic hydrolysis of pretreated redcedar was performed usingACCELLERASE® 1500 cellulase enzyme complex (Genencor Inc., Palo Alto,Calif., USA). The final glucose concentration was 73.8 g/L after 48 h ofhydrolysis, with a conversion efficiency of 88.6%. The hydrolyzate alsocontained 1.19±0.02 g/L cellobiose, 2.06±0.02 g/L xylose, 0.22±0.01 g/Lgalactose, and 0.91±0.01 g/L combined arabinose and mannoseconcentrations. The pH of the hydrolyzate decreased from 4.85 to 4.76during the hydrolysis.

Detoxification was used to remove inhibitors and the soluble lignin fromthe redcedar hydrolyzate before ABE fermentation. The soluble lignincontent (SLC) of the hydrolyzate prior to detoxification was 8.59 g/L.After detoxification with activated carbon, the SLC of the detoxifiedhydrolyzate was reduced to 2.69 g/L. The hydrolyzate was then filteredthrough a sterile filtration system followed by measuring the SLC of thesterile hydrolyzate. The SLC of the filtered hydrolyzate was 0.74 g/L.Detoxification and filter sterilization reduced the SLC of thehydrolyzate by 91%. In a previous study, the SLC of switchgrass wasreduced by 97% after detoxification and filter sterilization (Liu etal., 2015a). It was reported that a SLC above 1.77 g/L inhibited ABEfermentation (Wang and Chen, 2011). In the present study, glucoseconcentration of the filter sterile hydrolyzate was 70 g/L, which wasdiluted to 60 g/L in Medium II.

Sodium Bisulfite used in the pretreatment has an antibacterial effectwhich inhibited the bacterial growth in ABE fermentation (Liu et al.,2015b). Therefore, it is important to measure the bisulfite levels ofthe hydrolyzate after the biomass pretreatment process and prior to thefermentation. A reduction in bisulfate concentration in the hydrolyzatefrom 160 to 20 ppm was achieved after detoxification with powderedactivated carbon.

Co-Fermentation of Detoxified Redcedar Hydrolyzate and Generated CO₂ andH₂

Growth and product profiles of the co-fermentation in Medium II(detoxified redcedar hydrolyzate) in Fermentor A using C. acetobutylicumand CO₂ and H₂ in Fermentor B by C. ragsdalei are shown in FIG. 5 .Unlike ABE fermentation in Medium I (pure sugar) as shown in FIG. 3 ,Panel A, the maximum OD in the detoxified hydrolyzate Medium II inFermentor A was 4.2 units (FIG. 5 , Panel A). Lower growth of C.acetobutylicum was also reported in an earlier study with redcedarhydrolyzate compared to pure glucose medium (Liu et al., 2015b).However, comparable product profiles were obtained in Fermentor A usingMedium I and Medium II (FIGS. 3 and 5). Similar to ABE fermentation inMedium I (pure sugar), pH of Medium II in Fermentor A dropped from 6.4to 5.6 (FIG. 5 , Panel A). At the end of 72 h of fermentation, theresidual glucose concentration was 11.4 g/L (FIG. 5 , Panel B). Thehighest butanol and the total ABE production were 8.9 and 13.2 g/L,respectively (FIG. 5 , Panel C). The total acid production in FermentorA was 2.9 g/L.

Growth, pH, gas consumption/production, and product profiles of C.ragsdalei in Fermentor B are shown in FIG. 6 . The maximum OD inFermentor B was 0.6, and the pH was changed between 5.4 and 4.8.However, ethanol produced from H₂ and CO₂ generated from Medium II by C.ragsdalei in Fermentor B was about 0.9 g/L, which is lower compared toethanol produced when Medium I was used in Fermentor A (FIG. 4 , PanelC; and FIG. 6 , Panel C). However, slightly more acid was produced by C.ragsdalei in Fermentor B when Medium II was used. C. ragsdalei seems tobe slightly inhibited when Medium II was used in Fermentor A. The use ofco-fermentation has shown 7% and 182% improvements in ABE and totalorganic acid yields, respectively, due to additional ethanol and aceticacid produced from H₂ and CO₂.

The use of higher initial C. ragsdalei cell concentration in Fermentor Bcan further enhance gas conversion and production of more ethanol whenMedium II is used. FIG. 6 , Panel B shows changes in CO₂, H₂, and CO inheadspace of the co-fermentation system. Results showed some CO₂consumption between 48 to 72 h with overall accumulation of CO₂.However, similar to the Medium I experiment of Example 1, more than 95%of the initially added CO was utilized during the first 48 h of thefermentation. In addition, less H₂ consumption was noticed in FermentorB, which indicates inhibition of C. ragsdalei when Medium II containingredcedar hydrolyzate was used, which warrants further investigation.

The results clearly demonstrated the benefits of co-fermentation ofsugars and gases in increasing product yields, while utilizing typicallywasted gas streams. This novel process can be used in a multiple reactorsystem or a single reactor system, with various fermentation modes, andwith various other microorganisms to enhance product yields and toreduce emissions of CO₂. This novel process can be used to increaserevenues of various processes related to food, agriculture,pharmaceutical, environmental, and energy industries.

Example 3—Effects of Inoculum Preparation Methods on Conversion of CO₂and H₂ by the Co-Fermentation Process

Microorganisms and Inoculum Preparation

The inoculum preparation technique affects growth and product formationduring the conversion of CO₂ and H₂ gases in the co-fermentationprocesses of the present disclosure. In this Example, the conversion ofCO₂ and H₂ gases was performed using Clostridium ragsdalei, Clostridiumcarboxidivorans, and Clostridium muellerianum prepared by two differentinoculum methods. The first method used inoculum of each microorganismprepared with syngas Mix II containing 40% CO, 30% CO₂, and 30% H₂, byvolume. The second method used inoculum of each microorganism preparedwith gas Mix III containing 20% CO₂, 60% H₂, and 20% N₂, by volume.

Fermentation Medium

The three microorganisms were pre-cultured on standard medium thatcontained (per liter): 0.5 g yeast extract, 25 mL mineral stocksolution, 10 mL vitamin stock solution, 10 mL trace metals stocksolution, 10 g N-morpholinoethane-sulfonic acid (MES) buffer, 10 mL of4% cysteine sulfide solution, and 1 mL of 0.1% resazurin solution. Themineral stock solution contained (per liter): 100 g ammonium chloride,20 g magnesium sulfate, 10 g potassium chloride, 10 g potassiumphosphate monobasic, and 4 g calcium chloride. The vitamin stocksolution contained (per liter): 10 mg MESNA (2-mercaptoethanesulfonicacid sodium salt), 10 mg pyridoxine, 5 mg p-(4)-aminobenzoic acid, 5 mgcalcium pantothenate, 5 mg nicotinic acid, 5 mg riboflavin, 5 mgthiamine, 5 mg thioctic acid, 5 mg vitamin B12, 2 mg d-biotin, and 2 mgfolic acid. The trace metal stock solution contained (per liter): 2.0 gnitrilotriacetic acid, 1.0 g manganese sulfate, 1.0 g zinc sulfate, 0.8g ferrous ammonium sulfate, 0.2 g cobalt chloride, 0.2 g nickelchloride, 0.2 g sodium tungstate, 0.1 g sodium selenate, and 0.02 gsodium molybdate. The pH of the medium was adjusted during preparationto 6.0 using 5N KOH solution.

Inoculum Preparation Method 1

The first inoculum preparation method of each microorganism used thestandard medium with syngas Mix II containing 40% CO, 30% CO₂, and 30%H₂, by volume. Pre-culturing of each microorganism was performed inserum bottles containing standard medium (40 mL working volume) and 10mL of seed culture. Then, the culture bottles were flushed for 3 minwith synthesis gas (syngas) Mix I, which contained 20% CO, 15% CO₂, 5%H₂, and 60% N₂, by volume. The bottles were pressurized to 142.6 kPa(abs) with syngas Mix I and placed in a slant position in a warm room(37° C.) without agitation. After three days and at the beginning ofday-4, the headspace pressure was measured, the culture was fed syngasMix II containing 40% CO, 30% CO₂, and 30% H₂, by volume, to a pressureof 101.3 kPa (abs), and bottles were placed on a shaker at 125 rpm and37° C. At the beginning of day-5, the headspace pressure along with theoptical density (OD) and pH of the culture were measured. The pH of theculture was adjusted to 5.1-5.2 with 10% ammonium hydroxide if it wasbelow 5.0. Then, the bottles were pressurized to 142.6 kPa (abs) withsyngas Mix II and placed on a shaker at 125 rpm and 37° C. At thebeginning of day-6, the headspace pressure along with the opticaldensity (OD) and pH of the culture were measured. The pH of the culturewas adjusted to 5.1-5.2 with 10% ammonium hydroxide, if it was below5.0. The OD at the beginning of day-6 can reach between 0.5 to 0.6,which is good for transfer to another inoculum medium or a productionmedium. If the OD is below 0.5, the bottles were pressurized again withsyngas Mix II to 170.2 kPa (abs) and placed on a shaker at 125 rpm and37° C. to keep the inoculum for another day to allow OD to reach about0.6 before transferring it to inoculum medium or production medium. Thisculture was used to inoculate the gas fermentors.

Inoculum Preparation Method 2

The second inoculum preparation method of each microorganism used thestandard medium with gas Mix III containing 20% CO₂, 60% H₂, and 20% N₂,by volume. Pre-culturing of each microorganism was performed in serumbottles containing standard medium (40 mL working volume) and 10 mL ofseed culture. Then, the culture bottles were flushed for 3 min with gasMix III, which contained 20% CO₂, 60% H₂, and 20% N₂, by volume. Thebottles were pressurized to 170.2 kPa (abs) with gas Mix III and placedin a slant position in a warm room (37° C.) without agitation. At thebeginning of day-2, the bottles were placed on the shaker at 125 rpm and37° C. for 24 h. At the beginning of day-3, the headspace pressure alongwith the optical density (OD) and pH of the culture were measured. ThepH of the culture was adjusted to 5.1-5.2 with 10% ammonium hydroxide ifit was below 5.0. Then, the bottles were pressurized to 170.2 kPa (abs)with gas Mix III and placed on a shaker at 125 rpm and 37° C. At thebeginning of day-4, the headspace pressure along with the opticaldensity (OD) and pH of the culture were measured. The pH of the culturewas adjusted to 5.1-5.2 with 10% ammonium hydroxide if it was below 5.0.Then, the bottles were pressurized to 170.2 kPa (abs) with gas Mix IIIand placed on a shaker at 125 rpm and 37° C. At the beginning of day-5,the headspace pressure along with the optical density (OD) and pH of theculture were measured. The pH of the culture was adjusted to 5.1-5.2with 10% ammonium hydroxide if it was below 5.0. Then, the bottles werepressurized to 170.2 kPa (abs) with gas Mix III and placed on a shakerat 125 rpm and 37° C. At the beginning of day-6, the headspace pressurealong with the optical density (OD) and pH of the culture were measured.The pH of the culture was adjusted to 5.1-5.2 with 10% ammoniumhydroxide, if it was below 5.0. The OD at the beginning of day-6 reachedbetween 0.5 to 0.6, which is good for transfer to another inoculummedium or a production medium. If the OD is below 0.5, the bottles arepressurized again with gas Mix III to 170.2 kPa (abs) and placed on ashaker at 125 rpm and 37° C. to keep the inoculum for another day toallow OD to reach about 0.6 before transferring it to inoculum medium orproduction medium. This culture was used to inoculate the gasfermentors.

Reactor Configuration

The conversion of CO₂ and H₂ gases was performed in 250 mL serum bottleswith 40 mL working volume and 10 mL of seed culture at 37° C. and 125rpm. The headspace in the sealed bottles before inoculation contained20% CO₂, 60% H₂, and 20% N₂, by volume, at 101.3 kPa (abs). Then, 20%(v/v) of actively growing pre-culture of each microorganism wasseparately transferred into the serum bottles and fed with 20% CO₂, 60%H₂, and 20% N₂, by volume, at 170.1 kPa (abs). The bottles were fedevery 24 hours for 360 hours by gas Mix III containing 20% CO₂, 60% H₂,and 20% N₂, by volume, at 170.1 kPa (abs) and placed on a shaker at 125rpm and 37° C. Fermentations were performed in triplicates.

Analytical Methods

During fermentation, 1.5 mL liquid samples from each bottle wereaseptically taken at various times to determine cell growth, pH, andproduct profiles. The optical density (OD) of the fermentation mediumfrom the liquid samples from each bottle was determined using a UVspectrophotometer (UV-2100) at 600 nm for C. carboxidivorans and C.muellerianum and at 660 nm for C. ragsdalei. Solvents, ketones, andorganic acids produced were measured using GC-FID (GC 6890, AgilentTechnologies, Wilmington, Del., USA) as previously described (Liu etal., 2012; and Ramachandriya et al., 2013). The gas samples from theheadspace of each bottle was analyzed using GC-TCD (Liu et al., 2012).

Results and Discussion

Growth and product profiles of the conversion of CO₂ and H₂ gases usinginoculum methods 1 and 2 by C. ragsdalei, C. carboxidivorans, and C.muellerianum are shown in FIG. 7 . The growth profiles of the threemicroorganisms on CO₂ and H₂ were similar using inoculum method 1. Thehighest OD was achieved by C. muellerianum using inoculum method 1.However, C. ragsdalei with inoculum method 2 grew on CO₂ and H₂ to thehighest OD compared to C. carboxidivorans, and C. muellerianum. Thegrowth of C. carboxidivorans on CO₂ and H₂ was slower than C. ragsdaleiand C. muellerianum with inoculum method 2. There seems to be a laggrowth phase with inoculum method 2, especially with C. muellerianum.This clearly showed that the inoculum preparation method has a bigeffect on growth profiles. Inoculum method 1 is more favorable forgrowth on CO₂ and H₂ than inoculum method 2.

C. carboxidivorans and C. muellerianum produced 1.2-fold more aceticacid from CO₂ and H₂ using inoculum method 1 compared to inoculum method2 (FIG. 7 ). However, C. ragsdalei produced 1.9-fold more acetic acidfrom CO₂ and H₂ using inoculum method 2 compared to inoculum method 1.About 1.9- and 4.1-fold more acetic acid was produced from CO₂ and H₂ byC. ragsdalei compared to C. carboxidivorans, and C. muellerianum usinginoculum method 2. C. muellerianum made the lowest amount of acetic acidfrom CO₂ and H₂ using either inoculum methods. Inoculum method 2 is morefavorable for acetic acid production from CO₂ and H₂ by C. ragsdaleithan inoculum method 1. Also, inoculum method 1 is more favorable foracetic acid production from CO₂ and H₂ by C. carboxidivorans and C.muellerianum than inoculum method 2.

Over two-fold more ethanol was produced from CO₂ and H₂ by C. ragsdalei,C. carboxidivorans, and C. muellerianum using inoculum method 1 comparedto inoculum method 2 (FIG. 7 ). C. ragsdalei, C. carboxidivorans, and C.muellerianum produced similar amounts of ethanol from CO₂ and H₂ at 360h using inoculum method 1. However, C. muellerianum produced moreethanol from CO₂ and H₂ at 360 h using inoculum method 2 than C.ragsdalei and C. carboxidivorans. C. ragsdalei did not make any ethanolfrom CO₂ and H₂ using inoculum method 2. Inoculum method 1 is morefavorable for ethanol production from CO₂ and H₂ than inoculum method 2.

C. muellerianum produced 2-fold more butyric acid from CO₂ and H₂ usinginoculum method 1 compared to inoculum method 2 (FIG. 8 ). However, C.carboxidivorans only produced butyric acid from CO₂ and H₂ usinginoculum method 1. C. muellerianum produced 3.6-fold more butyric acidfrom CO₂ and H₂ using inoculum method 1. C. ragsdalei did not producedbutyric acid. Inoculum method 1 is more favorable for butyric acidproduction from CO₂ and H₂ than inoculum method 2.

C. muellerianum produced 4.2-fold more butanol from CO₂ and H₂ usinginoculum method 1 compared to inoculum method 2 (FIG. 8 ). However, C.carboxidivorans only produced butanol from CO₂ and H₂ using inoculummethod 1. C. muellerianum produced 7.5-fold more butanol from CO₂ and H₂using inoculum method 1. C. ragsdalei did not produced butanol. Inoculummethod 1 is more favorable for butanol production from CO₂ and H₂ thaninoculum method 2.

C. muellerianum produced 3.7-fold more hexanoic acid from CO₂ and H₂using inoculum method 1 compared to inoculum method 2 (FIG. 9 ). C.muellerianum only produced hexanol from CO₂ and H₂ using inoculummethod 1. C. ragsdalei and C. carboxidivorans did not produced hexanoicacid or hexanol. Inoculum method 1 is more favorable for hexanoic acidand hexanol production from CO₂ and H₂ than inoculum method 2.

Similar amounts of CO₂ were utilized by C. ragsdalei and C.carboxidivorans using inoculum method 1 (FIG. 10 ). However, C.ragsdalei consumed 1.6-fold more CO₂ than C. carboxidivorans usinginoculum method 2. C. ragsdalei consumed 1.2- and 2.4-fold more CO₂ thanC. muellerianum using inoculum method 1 and inoculum method 2,respectively. C. ragsdalei consumed about 1.4-fold more H₂ compared toC. carboxidivorans, and C. muellerianum using both inoculum method 1 andinoculum method 2. Generally, more CO₂ and H₂ were consumed by themicroorganisms using inoculum method 1 compared to inoculum method 2.Inoculum method 1 is more favorable for CO₂ and H₂ consumption comparedto inoculum method 2.

Based on the above, it was determined that Inoculum method 1 usingsyngas Mix II containing 40% CO, 30% CO₂, and 30% H₂ (by volume)improved growth, gas consumption, and production of alcohols such asethanol, butanol, hexanol, as well as fatty acids such as acetic acid,butyric acid, and hexanoic acid compared to inoculum method 2 with gasMix III containing 20% CO₂, 60% H₂, and 20% N₂ (by volume). In addition,inoculum method 1 reduced the lag phase for growth and productioncompared with inoculum method 2. Also, the presence of carbon monoxideduring inoculum preparation improved microbial activities and productformation during conversion of CO₂ and H₂ gases.

Thus, in accordance with the present disclosure, there have beenprovided compositions, as well as methods of producing and using same,which fully satisfy the objectives and advantages set forth hereinabove.Although the present disclosure has been described in conjunction withthe specific drawings, experimentation, results, and language set forthhereinabove, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art. Accordingly, itis intended to embrace all such alternatives, modifications, andvariations that fall within the spirit and broad scope of the presentdisclosure.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference. In addition, thefollowing is not intended to be an Information Disclosure Statement;rather, an Information Disclosure Statement in accordance with theprovisions of 37 CFR § 1.97 will be submitted separately.

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1. A method of biocatalytic conversion that utilizes a co-fermentationprocess for sugar and gaseous substrates, the method comprising thesteps of: (i) contacting at least one fermentation medium with at leastone first microorganism in a first reactor, wherein the at least onefermentation medium comprises at least one sugar substrate, and whereinthe at least one first microorganism converts the at least one sugarsubstrate into at least one of acetone, butanol, ethanol, isopropanol,acetic acid, and butyric acid, and wherein a gaseous substratecomprising CO₂ and H₂ gases is produced during the fermentation process;(ii) preparing an inoculum preparation of at least one secondmicroorganism by pre-culturing the at least one second microorganism inthe presence of a gas mixture comprising at least three gases selectedfrom the group consisting of carbon monoxide, carbon dioxide, hydrogen,and nitrogen; (iii) inoculating a second reactor with the inoculumpreparation of the at least one second microorganism; and (iv) feedingthe gaseous substrate produced in the first reactor into the secondreactor, the second reactor comprising at least one medium containingthe at least one second microorganism, wherein the at least one secondmicroorganism converts CO₂ and H₂ gases of the gaseous substrateproduced in the first reactor and fed into the second reactor into atleast one of an alcohol and an organic acid; wherein a substrateproduced in the second reactor is not fed into the first reactor; andwherein the second reactor has a volume that is larger than a volume ofthe first reactor.
 2. The method of claim 1, wherein the gas mixture of(ii) comprises at least three of: carbon monoxide gas in a range of fromabout 20% to about 40% by volume; carbon dioxide gas in a range of fromabout 15% to about 30% by volume; hydrogen gas in a range of from about5% to about 60% by volume; and nitrogen gas in a range of from about 20%to about 60% by volume.
 3. The method of claim 2, wherein the gasmixture comprises about 40% CO, about 30% CO₂, and about 30% H₂ byvolume.
 4. The method of claim 1, wherein each of the first and secondmicroorganisms comprises one or more species of microorganisms, whereineach species is from a genus selected from the group consisting ofClostridium, Butyribacterium, Eubacterium, Moorella, Acetobacterium,Enterobacter, Bacillus, Anaerobaculum, Alkalibaculum, and combinationsthereof, and wherein at least one of: the first microorganism comprisesat least one of Clostridium acetobutylicum, Bacillus firmus,Anaerobaculum hydrogeniformans, and Clostridium beijerinckii; and thesecond microorganism comprises at least one of Clostridium ragsdalei,Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridiumljungdahlii, Clostridium muellerianum, and Alkalibaculum bacchi. 5.(canceled)
 6. The method of claim 5, wherein at least one of: the atleast one first microorganism comprises Clostridium acetobutylicum ATCC824; the at least one second microorganism comprises Clostridiumragsdalei P11, and wherein ethanol, acetic acid, and isopropanol areproduced in the second reactor containing the at least one secondmicroorganism; the at least one second microorganism comprisesClostridium carboxidivorans, and wherein ethanol, butanol, hexanol,acetic acid, butyric acid, and hexanoic acid are produced in the secondreactor containing the at least one second microorganism; and/or the atleast one second microorganism comprises Clostridium muellerianum, andwherein ethanol, butanol, hexanol, acetic acid, butyric acid, andhexanoic acid are produced in the second reactor containing the at leastone second microorganism.
 7. The method of claim 1, wherein at least oneof: the at least one sugar substrate present in the at least onefermentation medium in the first reactor is selected from the groupconsisting of glucose, fructose, sucrose, xylose, galactose, arabinose,mannose, a starch, cellulose, hemicellulose, other carbohydrates,glucan, xylan, galactan, mannan, cellobiose, lignocellulosic biomass,and combinations thereof; the at least one fermentation medium in thefirst reactor contains a feedstock selected from the group consisting ofswitchgrass, forage sorghum, grassy materials, redcedar, woodymaterials, and combinations thereof, and wherein the method furthercomprises the step of pretreating and hydrolyzing the feedstock prior tocontact with the at least one first microorganism; and/or the method isfurther defined as producing at least one alcohol, at least one ketone,and at least one organic acid. 8-9. (canceled)
 10. The method of claim1, wherein at least one of: each of the first and second reactors ismaintained at a temperature in a range of from about 20° C. to about 45°C., and a pH of the at least one fermentation medium in the firstreactor is maintained in a range of from about 4 to about 7.5; the stepof feeding the gaseous substrate produced in the first reactor into thesecond reactor is further defined as feeding the gaseous substrate froma headspace of the first reactor into a headspace of the second reactor;and/or the step of feeding the gaseous substrate produced in the firstreactor into the second reactor is further defined as bubbling thegaseous substrate into the at least one medium of the second reactor;and/or a ratio of first reactor volume to second reactor volume is in arange of from about 1:2 to about 1:10.
 11. (canceled)
 12. The method ofclaim 10, wherein the ratio of first reactor volume to second reactorvolume is about 1:4.
 13. The method of claim 1, further comprising atleast one step selected from: the step of feeding additional carbonmonoxide (CO) and/or H₂ gas into the second reactor containing the atleast one second microorganism; the step of filling a headspace of thefirst reactor with a CO₂ concentration in a range of from about 40% toabout 100% prior to inoculation with the at least one firstmicroorganism; and/or a ratio of CO₂ to H₂ in the second reactor is in arange of from about 1:4 to about 4:1. 14-15. (canceled)
 16. Abiocatalytic conversion system that utilizes a co-fermentation processfor sugar and gaseous substrates, the biocatalytic conversion systemcomprising: a first reactor comprising at least one fermentation mediumcontaining at least one first microorganism, wherein the at least onefermentation medium comprises at least one sugar substrate, and whereinthe at least one first microorganism comprises a sugar fermentingspecies that converts sugars into at least one of acetone, butanol,ethanol, isopropanol, acetic acid, and butyric acid, and wherein agaseous substrate comprising CO₂ and H₂ gases is produced during thefermentation process; a second reactor comprising at least one mediumcontaining at least one second microorganism, wherein the at least onesecond microorganism comprises a gas fermenting species that convertsCO₂ and H₂ gases into at least one of an alcohol and an organic acid;and a gas line connecting the first reactor to the second reactor forfeeding the gaseous substrate produced in the first reactor into thesecond reactor; wherein the biocatalytic conversion system is absent asecond line for feeding a substrate produced in the second reactor intothe first reactor; and wherein a ratio of first reactor volume to secondreactor volume is in a range of from about 1:2 to about 1:10.
 17. Thebiocatalytic conversion system of claim 16, wherein each of the firstand second microorganisms comprises one or more species ofmicroorganisms, and wherein each species is from a genus selected fromthe group consisting of Clostridium, Butyribacterium, Eubacterium,Moorella, Acetobacterium, Enterobacter, Bacillus, Anaerobaculum,Alkalibaculum, and combinations thereof, and wherein at least one of:the first microorganism comprises at least one of Clostridiumacetobutylicum, Bacillus firmus, Anaerobaculum hydrogeniformans, andClostridium beijerinckii; and the second microorganism comprises atleast one of Clostridium ragsdalei, Clostridium autoethanogenum,Clostridium carboxidivorans, Clostridium ljungdahlii, Clostridiummuellerianum, and Alkalibaculum bacchi.
 18. (canceled)
 19. Thebiocatalytic conversion system of claim 18, wherein at least one of: theat least one first microorganism comprises Clostridium acetobutylicumATCC 824; the at least one second microorganism comprises Clostridiumragsdalei P11, and wherein ethanol, acetic acid, and isopropanol areproduced in the second reactor containing the at least one secondmicroorganism; the at least one second microorganism comprisesClostridium carboxidivorans, and wherein ethanol, butanol, hexanol,acetic acid, butyric acid, and hexanoic acid are produced in the secondreactor containing the at least one second microorganism; and/or the atleast one second microorganism comprises Clostridium muellerianum, andwherein ethanol, butanol, hexanol, acetic acid, butyric acid, andhexanoic acid are produced in the second reactor containing the at leastone second microorganism.
 20. The biocatalytic conversion system ofclaim 16, wherein at least one of: the at least one sugar substratepresent in the at least one fermentation medium in the first reactor isselected from the group consisting of glucose, fructose, sucrose,xylose, galactose, arabinose, mannose, a starch, cellulose,hemicellulose, other carbohydrates, glucan, xylan, galactan, mannan,cellobiose, lignocellulosic biomass, and combinations thereof; and/orthe at least one fermentation medium in the first reactor contains afeedstock selected from the group consisting of switchgrass, foragesorghum, grassy materials, redcedar, woody materials, and combinationsthereof, and wherein the feedstock has been pretreated and hydrolyzedprior to placement of the feedstock in the at least one fermentationmedium.
 21. (canceled)
 22. The biocatalytic conversion system of claim16, wherein at least one of: the gas line connects a headspace of thefirst reactor to a headspace of the second reactor; each of the firstand second reactors is maintained at a temperature in a range of fromabout 20° C. to about 45° C., and a pH of the at least one fermentationmedium in the first reactor is maintained in a range of from about 4 toabout 7.5; external carbon monoxide (CO) and/or H₂ gas is fed into thesecond reactor containing the at least one second microorganism; theratio of first reactor volume to second reactor volume is about 1:4;and/or a headspace of the first reactor has a CO₂ concentration in arange of from about 40% to about 100% prior to inoculation with the atleast one first microorganism. 23-26. (canceled)
 27. The biocatalyticconversion system of claim 22, wherein the CO₂ concentration is in arange of from about 40% to about 60%.
 28. A biocatalytic conversionsystem that utilizes a co-fermentation process for sugar and gaseoussubstrates, the biocatalytic conversion system comprising: a firstreactor comprising at least one fermentation medium containing at leastone first microorganism, wherein the at least one fermentation mediumcomprises at least one sugar substrate, and wherein the at least onefirst microorganism comprises a sugar fermenting species, wherein thesugar fermenting species is capable of converting sugars into at leastone of acetone, butanol, ethanol, isopropanol, acetic acid, and butyricacid, wherein the sugar fermenting species is capable of producing agaseous substrate comprising CO₂ and H₂ gases during the fermentationprocess, and wherein a headspace of the first reactor has a CO₂concentration in a range of from about 40% to about 100% prior toinoculation with the at least one first microorganism; a second reactorcomprising at least one medium containing at least one secondmicroorganism, wherein the at least one second microorganism comprises agas fermenting species, wherein the gas fermenting species is capable ofconverting CO₂ and H₂ gases into at least one of an alcohol and anorganic acid; and a gas line connecting the first reactor to the secondreactor for feeding the gaseous substrate produced in the first reactorinto the second reactor; wherein a ratio of first reactor volume tosecond reactor volume is in a range of from about 1:2 to about 1:10; andwherein the biocatalytic conversion system is absent a second line forfeeding a substrate produced in the second reactor into the firstreactor.
 29. The biocatalytic conversion system of claim 28, wherein atleast one of: the first microorganism comprises at least one ofClostridium acetobutylicum, Bacillus firmus, Anaerobaculumhydrogeniformans, and Clostridium beijerinckii; and the secondmicroorganism comprises at least one of Clostridium ragsdalei,Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridiumljungdahlii, Clostridium muellerianum, and Alkalibaculum bacchi.
 30. Thebiocatalytic conversion system of claim 29, wherein at least one of: theat least one first microorganism comprises Clostridium acetobutylicumATCC 824; the at least one second microorganism comprises Clostridiumragsdalei P11, and wherein ethanol, acetic acid, and isopropanol areproduced in the second reactor containing the at least one secondmicroorganism; the at least one second microorganism comprisesClostridium carboxidivorans, and wherein ethanol, butanol, hexanol,acetic acid, butyric acid, and hexanoic acid are produced in the secondreactor containing the at least one second microorganism; and/or the atleast one second microorganism comprises Clostridium muellerianum, andwherein ethanol, butanol, hexanol, acetic acid, butyric acid, andhexanoic acid are produced in the second reactor containing the at leastone second microorganism.
 31. The biocatalytic conversion system ofclaim 28, wherein at least one of: the at least one sugar substratepresent in the at least one fermentation medium in the first reactor isselected from the group consisting of glucose, fructose, sucrose,xylose, galactose, arabinose, mannose, a starch, cellulose,hemicellulose, other carbohydrates, glucan, xylan, galactan, mannan,cellobiose, lignocellulosic biomass, and combinations thereof; and/orthe at least one fermentation medium in the first reactor contains afeedstock selected from the group consisting of switchgrass, foragesorghum, grassy materials, redcedar, woody materials, and combinationsthereof, and wherein the feedstock has been pretreated and hydrolyzedprior to placement of the feedstock in the at least one fermentationmedium.
 32. (canceled)
 33. The biocatalytic conversion system of claim28, wherein at least one of: each of the first and second reactors ismaintained at a temperature in a range of from about 20° C. to about 45°C., and a pH of the at least one fermentation medium in the firstreactor is maintained in a range of from about 4 to about 7.5; externalcarbon monoxide (CO) and/or H₂ gas is fed into the second reactorcontaining the at least one second microorganism; and/or the ratio offirst reactor volume to second reactor volume is about 1:4. 34-56.(canceled)